Synthetic Molecular Probes of Endocytosis, Escape from Endosomes, and Protein-Protein Interactions

Abstract

Over the past several years, the Peterson group has developed synthetic mimics of cholesterol that rapidly incorporate into the plasma membrane of mammalian cells. These N-alkyl-3&beta-cholesterylamines cycle between the plasma membrane and early/recycling endosomes similar to natural cell surface receptors. Because of this unique activity, cholesterylamines have been linked to protein-binding and other motifs to generate artificial cell surface receptors. More recently, they have been conjugated to membrane disruptive peptides to generate synthetic agents that selectively permeabilize early endosomes and deliver cell-impermeable small molecules to the cytosol. Although this pioneering work has significant potential as a system to deliver cell-impermeable small molecule drugs and/or therapeutic agents, additional research is needed to understand the mechanism of action and develop therapeutic applications. To further evaluate this system, we first investigated the structure-activity relationships of cholesterylamines using fluorescent analogues designed to probe the mechanism of cellular uptake. This study produced probes with robust activity that suggests an unprecedented mechanism of cholesterol uptake on mammalian cell surfaces. Second, we investigated novel endosome disruptive peptides that provided insights into their mechanism of action. Additionally, we applied this knowledge to obtain proof-of-concept with antibody conjugates and endosome disruptive peptides as a new strategy to selectively deliver small molecules to tumor cells that overexpress specific cell surface receptors. Another project involves the development of a fluorescence-based method to detect protein-protein interactions in complex biological systems. This method utilizes a novel fluorinated fluorophore that undergoes proximity-driven exchange between lysine residues at the interface of a protein complex. Transfer of this fluorophore from donor to acceptor lysine residues produces a fluorescent protein partner that can be detected by gel electrophoresis or proteomics methods. Since lysine is prevalent at the interface of numerous protein complexes, this method may be useful to identify novel protein-protein interactions and/or factors that affect these interactions.